An important current goal in molecular biology is to understand how the synthesis and folding of proteins are coupled to each other. Although the understanding of events that occur as a protein is synthesized by the ribosome has been aided by high-resolution structures of many of the macromolecular components, only low-resolution views of the structure and dynamics of ribosome nascent-chain complexes (RNCs) are currently available. More importantly, essentially all studies of RNCs have been performed under conditions in which the RNC is stalled, i.e. under conditions in which its conformational dynamics are effectively at thermodynamic equilibrium. There is increasing evidence, however, that the fates of nascent proteins can depend significantly on the rate at which translation occurs, which implies that the partitioning of a newly synthesized protein between misfolded and native conformations is at least partly under kinetic control. There is, therefore, an urgent need for methods that can structurally characterize RNCs during active translation. Since experimental characterization is likely to remain an intractable problem it is proposed here to use molecular simulation methods instead. A plan of work is therefore outlined for developing a simulation framework that can accurately model coupled synthesis-folding events in the bacterial ribosome and that can fully define the role of its attached chaperone, trigger factor (TF), both in isolated monosomes and in models of complete polysomes. Three Specific Aims will be pursued. First, explicit-solvent molecular dynamics simulations will be used to determine the extent of trigger factor's conformational flexibility alone and in complex with the ribosome. These studies will establish the limits of TF's conformational adaptability in its functionally relevant states and will provide the basis for developing a realistic simulation model of TF-RNCs. Second, an accurate coarse-grained (CG) simulation model will be developed that allows the conformational behavior of stalled TF-RNC complexes to be directly modeled; properly parameterized, this model will enable a wide range of experimental observations on TF-RNCs to be rationalized at a truly structural level. Finally, the CG simulation model will be used to simulate cotranslational folding events in actively translating RNC complexes (with and without TF) in monosomes and polysomes. These latter studies will provide structural and dynamic pictures of nascent protein chains from the moment that they emerge from the ribosome's exit tunnel to the moment that they complete folding or misfolding in a way that is not achievable by conventional experimental methods. As such, the proposed studies are expected to greatly improve understanding of the factors that affect a nascent protein's propensity to fold or misfold during the course of its translation. PUBLIC HEALTH RELEVANCE: The proposed work is relevant to public health because it seeks to understand one of the potential origins of intracellular protein misfolding, an event now known to be implicated in a number of diseases. The work is relevant to the NIGMS's mission because it seeks to understand the basic science underpinning the cotranslational folding of proteins as they are synthesized by the ribosome, accounting also for the role(s) played by chaperones and assembly into polysomes.